C-terminally truncated, kidney-specific variants of the WNK4 kinase lack several sites that regulate its activity

Abstract

WNK lysine-deficient protein kinase 4 (WNK4) is an important regulator of renal salt handling. Mutations in its gene cause pseudohypoaldosteronism type II, mainly arising from overactivation of the renal Na/Cl cotransporter (NCC). In addition to full-length WNK4, we have observed faster migrating bands (between 95 and 130 kDa) in Western blots of kidney lysates. Therefore, we hypothesized that these could correspond to uncharacterized WNK4 variants. Here, using several WNK4 antibodies and WNK4 mice as controls, we showed that these bands indeed correspond to short WNK4 variants that are not observed in other tissue lysates. LC-MS/MS confirmed these bands as WNK4 variants that lack C-terminal segments. In HEK293 cells, truncation of WNK4's C terminus at several positions increased its kinase activity toward Ste20-related proline/alanine-rich kinase (SPAK), unless the truncated segment included the SPAK-binding site. Of note, this gain-of-function effect was due to the loss of a protein phosphatase 1 (PP1)-binding site in WNK4. Cotransfection with PP1 resulted in WNK4 dephosphorylation, an activity that was abrogated in the PP1-binding site WNK4 mutant. The electrophoretic mobility of the short variants of renal WNK4 suggested that they lack the SPAK-binding site and thus may not behave as constitutively active kinases toward SPAK. Finally, we show that at least one of the WNK4 short variants may be produced by proteolysis involving a Zn-dependent metalloprotease, as recombinant full-length WNK4 was cleaved when incubated with kidney lysate.

Zinc deficiency induces hypertension by promoting renal Na+ reabsorption

“…2, C, D, G, and H), indicating that ZnD stimulates phosphorylation of all expressed NCC. In support of this, a recent study implicated a Zn 2ϩdependent metalloprotease in regulation of WNK kinases, critical NCC regulators (16). Calcineurin is also a Zn 2ϩregulated phosphatase (17,23) linked to NCC phosphorylation (10).…”

“…Zn 2ϩ is an essential cofactor that influences the expression and activity of numerous enzymes, transcription factors, and regulatory proteins. Recently, it was suggested that a Zn 2ϩ -dependent metalloprotease may regulate with-no-lysine kinase (WNK) kinases, powerful regulators of NCC (16). However, a direct link between NCC and ZnD-induced BP dysregulation has not been investigated.…”

Calcium-Sensing Receptor and Regulation of WNK Kinases in the Kidney

“…In this context, the opposite effects of CaSR activation on NKCC2 vs. NCC deserve further clarification. Several C-terminally-truncated WNK4 variants have been identified in the kidney, including variants with increased, as well as decreased catalytic activity towards SPAK [94]. The described C-terminal truncations may eliminate the putative CaM-binding site (amino acids 1175-1194), thereby abrogating the inhibitory effects of [Ca 2+ ] i on WNK4 activity [69,94].…”

“…Several C-terminally-truncated WNK4 variants have been identified in the kidney, including variants with increased, as well as decreased catalytic activity towards SPAK [94]. The described C-terminal truncations may eliminate the putative CaM-binding site (amino acids 1175-1194), thereby abrogating the inhibitory effects of [Ca 2+ ] i on WNK4 activity [69,94]. Assuming distinct distribution of WNK4-variants between TAL and DCT, it is tempting to speculate that the truncated WNK4 variants lacking the inhibitory CaM-binding site but preserving the SPAK-binding motif are enriched in DCT.…”

“…Assuming distinct distribution of WNK4-variants between TAL and DCT, it is tempting to speculate that the truncated WNK4 variants lacking the inhibitory CaM-binding site but preserving the SPAK-binding motif are enriched in DCT. The CaSR-induced rise in [Ca 2+ ] i in DCT would then activate truncated WNK4 variants via Ca 2+ -binding to their acidic domain without competitive inhibitory effects mediated by C-terminal interaction with CaM [66,69,94]. Future studies using isolated rodent nephron segments are mandatory for resolving this issue.…”